Localization of aquaporin (AQP) water channels to either apical or basolateral membranes is important for various epithelial functions. We have established MDCK-II cell transfectants stably expressing AQP5 (RW5 cells) or AQP8 (RW8 cells). The expression of both AQPs was confirmed by the results of immunofluorescence microscopy and immunoblot analysis. When grown on polycarbonate filters, osmotically-obliged transepithelial water flow across RW5 and RW8 monolayers was similar to3-fold greater than that occurring across a monolayer of the parental cell line. Importantly, results of confocal immunofluorescence microscopy studies showed that AQP5 sorted to the apical membranes of RW5 cells. In contrast, AQP8 sorted to the basolateral membranes of RW8 cells. This is the first report of (i) stable epithelial cell cultures exhibiting a functional, polarized distribution of AQPs 5 and 8, and (ii) a basolateral localization of AQP8 in a polarized epithelial cell.